Bispecific Antibody With Prolonged Half-life And Enhanced Anti-tumor Effect

Abstract

Disclosed is a PEGylated bispecific antibody, comprising (a) an antigen-binding fragment (Fab), which has a light chain variable region (VL) and a light chain constant region (CL), a heavy chain Variable region (VH) and a part of the heavy chain constant region (CH1); (b) a single domain antigen binding fragment (VHH) fused to the C-terminus of the part of the heavy chain constant region (CH1) of the antigen binding fragment (Fab), and (c) Polyethylene glycol (PEG) fused to the C-terminus of the light chain constant region (CL) of the antigen-binding fragment (Fab). The PEGylated S-Fab (PEG-S-Fab) retained the binding specificity to both tumor cells and T cells. PEG-S-Fab enhanced the plasma stability and had a 12-fold increased half-life of S-Fab. PEG-S-Fab also had more potent tumor inhibiting efficacy in xenograft mouse model. Those data suggest that PEGylation can be an effective approach to enhance anti-tumor properties of bispecific antibodies.

Description

TECHNICAL FIELD

[0001] The present invention relates to bispecific antibodies, and in particular to PEGylated bispecific antibodies with prolonged half-life and enhanced anti-tumor effect.

BACKGROUND

[0002] Bispecific antibody has been developed as a powerful approach in cancer immunotherapy by engaging immune cells directly to the cancer cells. Bispecific antibodies have two different antigen binding sites with one recognizing the tumor cells and the other recognizing the immune cells, usually T cells or natural killer (NK) cells. Various bispecific antibody formats have been studied, including IgG based full-length formats (such as the complete bispecific antibody), single chain-based formats (including tandem single-chain variable fragments (ScFv)) and bispecific T-cell engager (BiTE).

[0003] In order to enhance the tumor tissue penetration of full-size IgG antibodies, and improve the stability of ScFv and BiTE, the bispecific single domain antibody-linked Fab (S-Fab) format was developed, which can bind to diverse epitopes and use prokaryotic expression systems. Derived from the natural camel heavy-chain only antibodies, single domain antibodies lack the first constant (CH1) domain and light chain, and are consequently referred to as nanobodies or VHHs. Nanobodies are small-sized and generally more stable than conventional scFv or BiTE, making them good specialty for constructing bispecific antibodies. In a previous study, a bispecfic S-Fab antibody was constructed by linking the single-domain nanobody anti-CEA to an CD3-Fab. The S-Fab exhibited excellent tumor cell killing activities in vitro. (L. Li, P. He, C. Zhou, L. Jing, B. Dong, S. Chen, N. Zhang, Y. Liu, J. Miao, Z. Wang, Q. Li, A novel bispecific antibody, S-Fab, induces potent cancer cell killing, Journal of immunotherapy, 38 (2015) 350-356).

[0004] However, Fab fragments (including S-Fab) still have short plasma half-lives as a result of rapid degradation in vivo, due to the lack of the constant regions (Fc), which makes them less ideal for long-term clinical use.

SUMMARY

[0005] In one aspect, provided is a PEGylated bispecific antibody (also called PEG-S-Fab herein), comprising (a) an antigen-binding fragment (Fab) having a light chain variable region (VL) and a light chain constant region (CL), as well as a heavy chain variable region (VH) and a part of a heavy chain constant region (CH1); (b) a single domain antigen-binding fragment (VHH) fused to the C-terminus of the part of the heavy chain constant region (CH1) of the antigen binding fragment (Fab); and (c) polyethylene glycol (PEG) fused to the C-terminus of the light chain constant region (CL) of the antigen-binding fragment (Fab). The molecular weight of PEG suitable for use in the present disclosure can be determined according to the routine test in the field. In some embodiments, the molecular weight of the polyethylene glycol is 10,000 to 30,000. In another embodiments, the molecular weight of the polyethylene glycol is 20,000.

[0006] The present invention also provides a pharmaceutical composition comprising the PEGylated bispecific antibody, and a pharmaceutically acceptable carrier or excipient. Correspondingly, the present invention provides a host cell comprising one or more polynucleotides encoding the bispecific antibody.

[0007] In another aspect, provided is an engineered bispecific antibody comprising (a) an antigen-binding fragment (Fab) having a light chain variable region (VL) and a light chain constant region (CL), a heavy chain variable region (VH) and a part of a heavy chain constant region (CH1); and (b) a single domain antigen binding fragment (VHH) fused to the C-terminus of the part of the heavy chain constant region (CH1) of the antigen binding fragment (Fab); wherein the C-terminus of the light chain constant region (CL) of the antigen-binding fragment (Fab) is engineered to have at least one cysteine residue. Correspondingly, the present invention provides a use of the engineered bispecific antibody in preparing the PEGylated bispecific antibody of the present invention.

[0008] Another aspect of the present invention provides a method of treating cancer, comprising contacting the PEGylated bispecific antibody of the present invention with cancer cells. Accordingly, the present invention provides a method of treating cancer in a subject, which comprises administering to the subject a therapeutically effective amount of the PEGylated bispecific antibody of the present invention.

[0009] Another aspect of the present invention provides a host cell comprising one or more polynucleotides encoding the bispecific antibody as described above. In some embodiments, the host cell is E. coli. The manipulation of polynucleotides involves knowledge and experimental operations in the fields of molecular biology, genetic engineering, protein engineering, etc., which are well known to those skilled in the art.

[0010] The inventors explored a thiol site-specific PEGylation to improve the half-life (a2) of S-Fab bispecific antibody. In this study, a functionalized 20 kDa linear methoxy PEG maleimide (MAL-PEG-OMe) was used to conjugate S-Fab. The PEGylated S-Fab (PEG-S-Fab) retained the binding specificity to both tumor cells and T cells. PEG-S-Fab enhanced the plasma stability and had a 12-fold increased half-life of S-Fab. PEG-S-Fab also had more potent tumor inhibiting efficacy in xenograft mouse model. Those data suggest that PEGylation can be an effective approach to enhance anti-tumor properties of bispecific antibodies.

BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 Expression and purification of S-Fab from E. coli. A, The bacterial S-Fab expression constructs contain a pelB signal sequence, an anti-CD3 (human UTCH1 clone) VH (or VL) and CH (CL), and an anti-CEA-VHH. To facilitate antibody detection and purification, a flag-tag and a his6-tag were added to the C-terminal end of heavy and light chain, respectively; B, Schematic presentation of S-Fab after co-expression; C, Coomassie blue-stained SDS-PAGE of the purified S-Fab after the two-step purification; D, Gel filtration analysis showed that the molecular weight of S-Fab was approximately 130 kD.

[0012] FIG. 2 PEGylation of bispecific S-Fab using 20 kDa linear methoxy PEG maleimide (MAL-PEG-OMe). S-Fab reacted with the functionalized PEG at a series of ratios (0:1, 10:1, 20:1, 40:1 and 60:1) mol equiv of PEG:S-Fab and shaking at 22.degree. C. for 2 hrs, the samples were then examined with SDS-PAGE (5 ul/sample). A. Coomassie blue staining for the S-Fab with different molar ratio of PEG:S-Fab in PEGylation process. B. Barium iodide complex dye staining for the S-Fab with different molar ratios of PEG:S-Fab. C. Western blot using anti-flag antibody to detect the VH--CH-CEA chain. D. Western blot using anti-his6 antibody to detect the VL-CL chain. Note: 0, 10, 20, 40 and 60 represent as 0:1, 10:1, 20:1, 40:1 and 60:1 for the molar ratio of PEG:Fab.

[0013] FIG. 3 Purification of PEG-S-Fab. The gel filtration was used to fraction the PEGylation mixture (Fractions 1, 2, 3). A. Chromatogram of PEG-S-Fab. B. Coomassie blue staining for the fractions of PEG-S-Fab purification. C. Barium iodide complex staining for the fractions of PEG-S-Fab purification.

[0014] FIG. 4 PEG-S-Fab can bind CEA on tumor cells and CD3.sup.+ on T cells. Flow cytometry analysis with PEG-S-Fab and S-Fab was performed on CEA-positive LS174T cells (A), CEA-negative SKOV3 cells (B), and CD3.sup.+ T cells (C). Positive control anti-CD3 antibody OKT3 was used for T cell flow cytometry. Confocal microcopy of immunofluorescence staining of S-Fab (D) and PEG-S-Fab (E) in LS174T cells (upper panel) and SKOV3 cells (lower panel), respectively. The bar scale represents 30 .mu.m.

[0015] FIG. 5 PEG-S-Fab has potent specific cytotoxicity against tumor cells. A. The cytotoxic assay using CEA-positive LS174T cells. B. The cytotoxic assay using CEA-negative SKOV3 cells. The cytotoxic assays were performed as described in section 2.8. Different concentrations of S-Fab or PEG-S-Fab were incubated with tumor cells and effector T cells (E/T=10). All data are showed as the mean of triplicates, with error bars representing the SD.

[0016] FIG. 6 Pharmacokinetic and stability analysis of serum concentrations of PEG-S-Fab and S-Fab. A. Standard curve for the quantified ELISA. B. The serum protein concentration-time curve after intravenous administration. (Note: Each data point was expressed as the mean.+-.SEM). C-D. Western blotting to detect the resulting S-Fab (C) and PEG-S-Fab (D) using anti-flag antibody. E-F. Western blotting to detect the resulting S-Fab (C) and PEG-S-Fab (D) using anti-his antibody. (Note: M, P--S--Fab and P represent as marker, PEG-S-Fab and plasma only, respectively).

[0017] FIG. 7 PEGylation of S-Fab induces more potent in vivo anti-tumor activity. B-NDG mice (n=6 per group) were subcutaneously engrafted with LS174T cells and human PBMCs. The mice were then treated with PBS alone, 0.3 nmol of S-Fab or S-Fab-PEG20K daily over six days. The data represent the average tumor volume of six mice. The error bars represent the SEM. Data were analyzed with two-way ANOVA analysis using GraphPad Prism 5 software, and *** represents as p<0.01 when compared the vehicle with S-Fab or PEG-S-Fab groups.

DETAILED DESCRIPTION OF THE INVENTION

Definition

[0018] It should be noted that the term "a" or "an" entity refers to one or more of that entity, for example, "a bispecific antibody" is understood to represent one or more bispecific antibodies. Likewise, the terms "a", "one or more" and "at least one" are used interchangeably herein.

[0019] The term "antibody" or "antigen-binding polypeptide" as used herein refers to a polypeptide or polypeptide complex that specifically recognizes and binds to one or more antigens. Antibodies are complete antibodies or any antigen-binding fragments or single chains thereof. Therefore, the term "antibody" includes any protein or peptide that contains at least a part of an immunoglobulin molecule that has the biological activity of binding to an antigen. Such examples include, but are not limited to, the complementarity determining region (CDR) of the heavy/light chain or its ligand binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, and the framework (FR) region or any part thereof, or at least part of a binding protein. The term antibody also encompasses polypeptides or polypeptide complexes that have antigen-binding ability once activated. In some examples, for example, certain immunoglobulin molecules derived from or based on the engineering of camelid immunoglobulins. The whole immunoglobulin molecule may only consist of heavy chains without light chains, see for example Hamers-Casterman et al., Nature 363:446-448 (1993).

[0020] The term "specific binding" or "specific to" generally means that the antibody binds to the epitope through its antigen binding domain, and the binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, when an antibody can bind to a specific epitope more rapidly via its antigen binding domain than to a random unrelated epitope, the antibody is said to "specifically bind" to that epitope. The term "specificity" is used to quantify the relative affinity of a specific antibody to a specific epitope. For example, for a given epitope, antibody "A" is considered to have a higher specificity than antibody "B", or antibody "A" binds to epitope "C" more specifically than it binds to related epitope "D".

[0021] The term "treatment" as used herein refers to a therapeutic or preventive approach in which a subject is prevented from or slowed down (alleviated) an undesirable pathological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (that is, no deterioration), delay or slowing of disease progression, improvement or alleviation of disease state, and alleviation (either locally or systematically), detectable or undetectable. The term "treatment" also refers to prolonged survival compared to expected survival if not receiving the treatment. Subject in need of treatment include those who already have a disease or condition, as well as those who tend to have the disease or condition or those who need to prevent the disease or condition.

[0022] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for which diagnosis, prognosis, or treatment is desired, especially a mammalian subject. Mammal subjects include humans, domestic animals, farm animals, zoo animals, sports animals or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, bovines, cows, and the like.

[0023] The term "patient in need of treatment" or "subject in need of treatment" includes subjects who can benefit from the administration of the antibody or composition of the present invention, such as mammalian subjects, for purposes of such as detection, diagnostic procedures and/or treatment.

[0024] As confirmed in the examples, an exemplary bispecific antibody is an antibody that targets two different antigens, one of which is present on tumor cells or microorganisms, and the other is on immune cells. When administered to an individual, the bispecific antibody specifically binds to tumor cells or microorganisms, and at the same time specifically binds to immune cells (such as cytotoxic cells). This dual binding can cause the bound tumor or microorganism to be killed by the host's immune system.

[0025] A "single domain antigen-binding fragment" or "single domain antibody fragment" or "VHH" is an antigen-binding fragment capable of binding to an antigen without being equipped with a light chain. VHH was originally isolated from a single domain antibody (sdAb) as a single antigen-binding fragment. The first known single domain antibody was isolated from camel (Hamers-Casterman et al., Nature 363:446-8 (1993)) and later from cartilaginous fish. Camels produce functional antibodies without light chains, and their single N-terminal domain (VHH) binds to antigen without domain pairing (see Harmsen and Haard, App Microbiol Biotechnol, 77:13-22 (2007) for review). Single domain antibodies do not include the CH1 domain which, in conventional antibodies, interacts with the light chain.

[0026] VHH contains four framework regions (FR1-FR4) constituting the core structure of an immunoglobulin domain and three complementarity determining regions (CDR1-CDR3) involved in antigen binding. Compared with the human VH domain, the VHH framework region shows high sequence homology (>80%) with the human VH domain. See Harmsen and Haard, 2007, which further describes: "the most characteristic feature of VHH lies in the amino acid substitutions at the four FR2 positions (positions 37, 44, 45 and 47; Kabat numbering), which are conserved and involved in hydrophobic interactions with the VL domain in conventional VH structures". VHH usually has different amino acids at these and other positions that are highly conserved in conventional VH (such as LeulISer, Val37Phe or Tyr, Gly44Glu, Leu45Arg or Cys, Trp47Gly).

[0027] Harmsen and Haard, 2007 also described: VHH CDR has some known characteristic features. For example, the N-terminal part of CDR1 is more variable than conventional antibodies. Moreover, certain VHHs have a next ended CDR3 which is usually stabilized by forming additional disulfide bonds with the cysteine in CDR1 or FR2, resulting in the CDR3 loop folding over the entire interface of the previous VL. The specific subfamily of llama VHH (VHH3) also contains an extended CDR3 which is stabilized by forming an additional disulfide bond width cysteine at position 50 of FR2.

[0028] Many single domain antibodies (sdAbs) are known in the art and can be easily prepared from animals such as camels. Based on these sdAbs, their VHH can be easily identified and prepared. Table 1 lists many non-limiting examples of VHH and sdAb. Therefore, in some embodiments, the present invention provides a polypeptide comprising each such disclosed sequence or its equivalent and a polynucleotide encoding each polypeptide.

TABLE-US-00001 TABLE 1 Exemplary single domain antigen binding fragments (VHH) and single domain antibodies (sdAb) 1. anti-CEA VHH (SEQ ID NO: 1) EVQLVESGGG FVQAGESLTL SCTSSTLTFT PYRMAWYRQA PGKQRDLVAD ISSGDGRTTN YADFAKGRFT ISRDNIKNTV FLRMTNLKPE DTAVYYCNTF VSFVGIARSW GQGTQVTVSS 2. anti-CD16 VHH (SEQ. ID NO: 2) EVQLVESGGG LVQPGGSLRL SCSFPGSIFS LTMGWYRQAP GKERELVTSA TEGGDTNYAD FVKGRFTISR DNARSIIYLQ MNSLKPEDTA VYYCYARTRN WG 3. anti-CD16 VHH (SEQ ID NO: 3) EVQLVESGGE LVQAGGSLRL SCAASGLTFS SYNMGWFRRA PGKEREFVAS ITWSGRDTFY ADSVKGRFTI SRDNAKNTVY LQMSSLKPED TAVYYCAANP WP 4. anti-CD16 VHH (SEQ ID NO: 4) EVQLVESGGG LVQEGESLTL SCVVAGSIFS FAMSWYRQAP GKERELVARI GSDDRVTYAD SVKGRFTISR DNIKRTAGLQ MNSLKPEDTA VYYCNAQTDL RD 5. anti-CD16 VHH (SEQ ID NO: 5) EVQLVESGGG LVQPGGSLTL SCVAAGSIFT FAMSWYRQAP RKERELVARI GTDDETMYKD SVKGRFTISR DNVKRTAGLQ MNNLKPEDTA VYYCNARTDY RD 6. anti-Her2 sdAb (SEQ ID NO: 6) QVQLVQSGGG LVQAGGSLRL SCAASGRTFS SYAMAWFRQA PGKEREFVAA ISWSGANIYV ADSVKGRFTI SRDNAKDTVY LQMNSLKPED TAVYYCAVKL GFAPVEERQY DYWGQGTQVT VSS 7. anti-EGFR1 VHH (SEQ ID NO: 7) MAEVQQASGG GLVQAGGSLR LSCAASGRTE TTSAIAWFRQ APGKEREFVA QISASGLGIN YSGTVKGRFT ISRDADKTTV YLQMNSLTPE DTAVYYCAAG FHYIAAIRRT TDFHFWGPGT LVTVSSGR 8. anti-F4 + ETEC bacteria VHH (SEQ ID NO: 8) QVQLQESGGG LVQAGGSLRL SCEASGNVDR IDAMGWERQA PGKQREFVGY ISEGGILNYG DEVKGRETIS RDNAKNTVYL QMSNLKSEDT GVYFCAASHW GTLLIKGTEH WGKGTQVTVS S 9. anti-PS2-8 VHH (SEQ ID NO: 9) EVQLVESGGG LVQAGGSLRL SCAASGRSFS RDAMGWFRQA PGKERDVVAA INLNGGRTYS ADSVKGPFTI SRDNDKNTVY LQMSNLKPED TAVYYCAARE GDVGLVSYKR SSNYPYWGQG TQVTVSS 10. anti-Huntavirus VHH (SEQ ID NO: 10) MAEVQLQASG GGLVQAGGSL RLSCAASGRT SSMYSMVWFR QAPGKEREFV AGIIWTSSLT YYADSLKGRF TISRDNAKNT VYLQMNSLKP EDTAIYYCAA DTKTGGGGYE YWGQVTVTVS S 11. anti-Huntavirus VHH (SEQ ID NO: 11) MAEVQLQASG GGLVQPGGSL RLSCAASGSI FSSDVMGWFR QAPGKERELV AFITDDGGTN YADSVKGRFT ISRDNAENTV SLQMNSLKPE DTAVYYCNAR YYSGGYRNYW GQVTVTVSS 12. anti-CD16 VHH (SEQ ID NO: 12) EVQLVESGGG LVQPGGSLRL SCSFPGSIFS LTMGWYRQAP GKERELVTSA TPGGDTNYAD FVKGRETISR DNARSIIYLQ MNSLKPEDTA VYYCYARTPN WGTVWGQGTQVTVSS 13. anti-CD3 VHH (SEQ ID NO: 13) GVQLQESGGG LVQAGGSLRL SCAASGRTFS NYHMGWFRQA PGKERELVAA ISGSGGSTYY TDSVFGRFTI SPNNAKNTMS LQMSNLKPED TGVYYCTTPT EKGSSIDYWG QGTQVTVSSG RYPYDVPDY

[0029] In some embodiments, the Fab or VHH (or VHH') fragment of the bispecific antibody has immunologic specificity to tumor antigens.

[0030] "Tumor antigen" is anantigenic substance produced in tumor cells, that is, it causes an immune response from the host. Tumor antigens can be used to identify, tumor cells and are potential candidates for cancer treatment. The normal protein in the body is not antigenic. However, certain proteins are produced or overexpressed during the tumorigenesis process and therefore appear to be "foreign" to the body. This can include normal proteins that well evade the immune system, proteins that are usually produced in very small amount, proteins that are usually only produced at specific developmental stages, or proteins whose structure is modified due to mutations.

[0031] Many tumor antigens are known in the art, and many new tumor antigens can be easily discovered through screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD 133, CEA, gpA33, mucin, TAG-72, CIX PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, .alpha.V.beta.3, .alpha.5.beta.1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

[0032] In some aspects, Fab or VHH fragments have specificity for proteins that are overexpressed on tumor cells compared to corresponding non-tumor cells. "Corresponding non-tumor cells" refers to non-tumor cells that have the same cell type as the cells from which the tumor cells are derived. It should be noted that such proteins are not necessarily different from tumor antigens. Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectal, breast, lung, pancreatic, and gastrointestinal cancers; mygulin receptor (HER-2, neu or c-erbB-2), which is usually overexpressed in breast, ovarian, rectal, lung, prostate and cervical cancers; epidermal growth factor receptor, which is overexpressed in a series of solid tumors (including breast cancer, head and neck cancer, non-small cell lung cancer and prostate cancer); asialoglycoprotein receptor; transferin receptor; serine protease inhibitor enzyme complex receptor expressed on liver cells; fibroblast growth factor receptor (FGFR) over-expressed on pancreatic ductal adenocarcinoma cells; vascular endothelial growth factor receptor (VEGFR) for anti-angiogenesis gene therapy; folate receptor selectively overexpressed in 90% of non-mucinous ovarian cancer; polysaccharide-protein complexes (glycocalyx) on cell surface; carbohydrate receptors; and polyimmunoglobulin receptors, which are beneficial for gene transfer to respiratory epithelial cells and are attractive for the treatment of lung diseases (such as cystic fibrosis).

[0033] In some aspects, the Fab portion includes: one or two amino acid sequences selected from SEQ ID NO: 14-25 (Table 2), or optionally with one or two or three insertions, deletions or substitutions.

TABLE-US-00002 TABLE 2 Examples of Fab sequences 1. anti-CD19Fab light chain-nucleic acid (SEQ ID NO: 14) GATATTCAGATGACTCAGAGCCCTTCCTCACTGTCCGCCAGCGTCGGGGATCGGGTCACT ATTACATGCAAAGCAAGCCAGAGCGTCGACTATGATGGGGACTCCTATCTGAACTGGTAC CAGCAGAAGCCAGGCAAAGCTCCCAAGCGGCTGATCTACGACGCATCAAATCTGGTGAGC GGCGTCCCTTCCAGATTCTCTGGGAGTGGATCAGGCACAGATTTTACCCTGACAATTAGC TCCCTGCAGCCTGAGGACTTCGCCACTTACTATTGCCAGCAGAGCACAGAAGATCCATGG ACTTTTGGCGGGGGAACCAAAGTGGAGATCAAGAGA (VL) ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCGACGGCTGAGCAAAGCAGACTACGAGAAA CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGT (CL) 2. anti-CD19Fab light chain - amino acid (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYLNWYQQKPGKAPKRLIYDASNLVS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTEDPWTFGGGTKVEIKR (VL) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CL) 3. anti-CD19Fab heavy chain-nucleic acid (SEQ ID NO: 16) CAGGTGCAGCTGGTCCAGTCCGGGGCAGAAGTGAAGAAACCAGGAGCCTCTGTGAAAGTC AGTTGTAAGGCTTCAGGCTATACCTTCATCTCTTACTGGATGAACTGGGTGAGACAGGCA CCAGGACAGGGACTGGAGTGGATGGGACAGATTTGGCCTGGCGATGGGGACACCAACTAT AATCAGCTGCTGAAGGATAAAGCAACTCTGACCGCCGACAAGAGCGCCTCCACCGCTTAC ATGGAGCTGTCTAGTCTGCGAAGCGAAGACACAGCTGTGTACTATTGCGCACGGAGAGAA ACCACAACTGTGGGCCGCTACTATTACGCAATGGATTACTGGGGACAGGGCACACTGGTG ACTGTCTCAAGC (VH) GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT (CH) 4. anti-CD19Fab heavy chain-amino acid (SEQ ID NO: 17) QVQLVQSGAEVKKPGASVKVSCKASGYTFISYWMNWVRQAPGQGLEWMGQIWPGDGDTNY NQLLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARPETTTVGRYYYAMDYWGQGTLV TVSS (VH) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (CH) 5. anti-CD20Fab light chain-nucleic acid (SEQ ID NO: 18) CAGATCGTGCTGAGCCAGAGCCCCGCCATCCTGAGCGCCAGCCCCGGCGAGAAGGTGACC ATGACCTGCAGGGCCAGCAGCAGCGTGAGCTACATCCACTGGTTCCAGCAGAAGCCCGGC AGCAGCCCCAAGCCCTGGATCTACGCCACCAGCAACCTGGCCAGCGGCGTGCCCGTGAGG TTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGGTGGAGGCCGAG GACGCCGCCACCTACTACTGCCAGCAGTGGACCAGCAACCCCCCCACCTTCGGCGGCGGC ACCAAGCTGGAGATCAAGAGG (VL) ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGAGGCTGAGCAAAGCAGACTACGAGAAA CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGT (CL) 6. anti-CD20Fab light chain-amino acid (SEQ ID NO: 19) QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVR FSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR (VL) TVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CL) 7. anti-CD20Fab heavy chain-nucleic acid (SEQ ID NO: 20) CAGGTGCAGCTGCAGCAGCCCGGCGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATG AGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACC CCCGGCAGGGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACCAGCTAC AACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTAC ATGCAGCTGAGCAGCCTGAGCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAGCACC TACTACGGCGGCGACTGGTACTTCAACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGC (VH) GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT (CH) 8. anti-CD20Fab heavy chain-amino acid (SEQ ID NO: 21) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSY NQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS (VH) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (CH) 9. anti-Her2Fab light chain-nucleic acid (SEQ ID NO: 2) GATATTCAGA TGACCCAGAG CCCGAGCAGC CTGTCCGCAA GCGTCGGCGA CCGCGTCACG ATTACCTGCC GTGCAAGCCA GGATGTCAAC ACCGCAGTGG CTTGGTATCA GCAAAAACCG GGTAAAGCGC CGAAACTGCT GATTTATAGT GCCTCCTTTC TGTACTCTGG CGTGCCGAGT CGTTTCTCAG GTTCGCGCAG CGGCACCGAT TTTACCCTGA CGATCAGCTC TCTGCAGCCG GAAGACTTCG CCACGTATTA CTGCCAGCAA CATTATACCA CGCCGCCGAC CTTTGGTCAA GGCACGAAAG TCGAAATTAA A (VL) ACGGTGGCTG CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA AGTAGAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGIAAC TCCCAGGAGA GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG GAGAGTGT (CL) 10. anti-Her2Fab light chain-amino acid (SEQ ID NO: 23) DIQMTQSPSSLSASVGDPVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTEGQGTKVEIK (VL) TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC (CL) 11. anti-Her2Fab heavy chain-nucleic acid (SEQ ID NO: 24) GAAGTGCAGC TGGTTGAATC TGGCGGTGGC CTGGTTCAAC CGGGTGGCTC CCTGCGTCTG TCATGTGCAG CATCGGGTTT CAACATTAAA GATACCTATA TCCACTGGGT TCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTCGCTCGC ATTTACCCGA CCAATGGTTA TACGCGTTAC GCGGATAGCG TGAAAGGCCG CTTCACCATC AGCGCAGACA CCTCTAAAAA CACGGCTTAT CTGCAGATGA ACTCGCTGCG TGCGGAAGAT ACGGCCGTTT ATTACTGTAG CCGCTGGGGT GGCGACGGCT TTTACGCAAT GGATTACTGG GGTCAAGGCA CGCTGGTCAC GGTCTCA (VH) GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA AGTT (CH) 12. anti-Her2Fab heavy chain-amino acid (SEQ ID NO: 25) EVQLVESGGGLVQGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS (VH) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (CH)

[0034] Any of the antibodies or polypeptides described above may further include additional polypeptides, for example, a signal peptide that directs the secretion of the encoded polypeptide, an antibody constant region as described herein, or other heterologous polypeptides as described herein.

[0035] Those of ordinary skill in the art will understand that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the natural binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specific protein may be similar, for example it has a certain percentage of identity to the starting sequence, for example, it may have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0036] In addition, conservative substitutions, deletions or insertions of nucleotides or amino acids can be made in the "non-essential" amino acid region. For example, a polypeptide or amino acid derived from a specific protein may be the same as the starting sequence, except for one or more single amino acid substitutions, insertions, or deletions (e.g., one, two, three, four, five, six, Seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions or deletions). In certain embodiments, compared to the starting sequence, the polypeptide or amino acid sequence derived from a particular protein has one to five, one to ten, or one to twenty individual amino acid substitutions, insertions, or deletions. In other embodiments, the antigen-binding polypeptides of the present invention may contain conservative amino acid substitutions.

[0037] In "conservative amino acid substitutions," one amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid residue families with similar side chains have been defined in the art, including basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), .beta. branched side chains (such as threonine, valine, isoleucine) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides are more suitable to be replaced by other amino acid residues from the same side chain family. In another embodiment, the amino acid chain may be replaced by a chain that is structurally similar but differs in the order or component of the side chain family members.

[0038] The table below provides non-limiting examples of conservative amino acid substitutions. The similarity score of 0 or higher in the table indicates conservative substitutions between two amino acids.

TABLE-US-00003 C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0039] In some embodiments, antibodies can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological effect modifiers, or pharmaceutical formulations.

[0040] These antibodies can be conjugated or fused to therapeutic agents, which may include detectable labels (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutic agents or diagnostic agents, cytotoxic agents (drugs or toxins), ultrasound enhancers, non-radioactive labels, their combinations, and other agents known in the art.

[0041] By coupling to a chemiluminescent compound to label the antibody, the antibody can be detected. Then, by detecting the presence of fluorescence (which occurs during the chemical reaction), the presence of chemiluminescent labeled antigen-binding peptides is determined. Examples of extremely useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0042] As mentioned above, the bispecific antibodies, vanants or derivatives thereof of the present invention can be used in certain methods of treatment and diagnosis related to cancer or infectious diseases.

Treatment and Therapy

[0043] The present invention further relates to antibody-based therapy, which involves administering the bispecific antibody of the present invention to patients, such as animals, mammals, and humans, to treat one or more diseases or symptoms described herein. The therapeutic composition of the present invention includes, but is not limited to, the antibody of the present invention (including the variants and derivatives thereof described herein) and nucleic acid or polynucleotide (including the variants and derivatives thereof described herein) encoding the antibody of the present invention.

[0044] The antibodies of the present invention can be used to treat, inhibit or prevent the following diseases, disorders, or conditions, including, for example, malignant diseases, disorders, or conditions (such as cancers) associated with increasing cell survival or inhibiting cell apoptosis. The cancers include but are not limited to follicular lymphoma, cancers with p53 mutations, and hormone-dependent tumors (including but not limited to colon cancer, heart tumors, pancreatic cancer, melanoma, retinoblastoma, malignant glioma, lung cancer, colorectal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenocarcinoma, breast cancer, prostate cancer, Kaposi's sarcoma); autoimmune disorders (such as multiple sclerosis, Sjogren syndrome. Grave disease. Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymvositis, systemic lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura and rheumatoid arthritis); and viral infections (such as herpes virus, poxvirus and adenovirus), inflammation, graf-versus-host disease (acute and/or chronic), acute graft rejection, and chronic graft rejection. The antigen-binding polypeptides, variants or derivatives thereof of the present invention are used to inhibit the development, evolution and/or metastasis of cancer, especially the cancers listed in the above or subsequent paragraphs.

[0045] The antibody of the present invention can also be used to treat infectious diseases caused by microorganisms or kill microorganisms by targeting microorganisms and immune cells to affect the elimination of microorganisms. In one aspect, the microorganism is a virus (including RNA and DNA viruses), gram-positive bacteria, gram-negative bacteria, protozoa, or fungi.

[0046] The specific dosage and treatment regimen for any particular patient will depend on many factors, including specific antigen-binding polypeptide, its variants or derivatives used, patient's age, weight, general health, gender, diet, time of administration, excretion rate, combination of drugs, and severity of the specific disease being treated. The physician's judgment on such factors falls within the judgment of those of ordinary skill in the art. The dosage will also be based on the individual patient being treated, route of administration, type of formulation, the characteristics of the composition used, the severity of the disease, and the desired effect. The dosage can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0047] Methods of administration of the bispecific antibodies and variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, for example, by infusion or bolus injection, or absorption through epithelial or mucosal protective layer (such as oral mucosa, rectum and intestinal mucosa, etc.); it can be used in combination with other biologically active preparations. Therefore, the pharmaceutical composition containing the antigen-binding polypeptide of the present invention can be administered via oral cavity, rectum, parenteral, vaginal, abdominal cavity, topically (such as by powder, ointment, drops or skin patch), buccal, or oral or nasal cavity.

[0048] The term "parenteral" as used herein refers to the mode of administration, which includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

[0049] Administration can be systemic or local. In addition, it is expected that the antibody of the present invention can be introduced into the central nervous system by any suitable route, including intracerebroventricular and intrathecal injection; intraventricular catheters, for example, connected to a reservoir (such as Ommaya reservoir), can be used to facilitate intraventricular injection. Pulmonary administration can also be used, for example by using inhalers or nebulizers and formulations with aerosols.

[0050] It may be desirable to apply the bispecific antibody or composition of the present invention locally to the area in need of treatment; this can be achieved, for example, but not limited to, local infusion during surgery, local application (for example, combined use with wound dressings), injections, catheters, suppositories, or implants (implants are porous, non-porous, non-permeable or gel-like materials, including films (such as silicone membranes) or fibers)). Preferably, when administering a protein (including an antibody) including the antibody of the present invention, care should be taken to use a material that does not adsorb the protein.

[0051] The effective amount of the antibody of the present invention in the treatment, inhibition and prevention of inflammation, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help identify the optimal dose range. The exact dosage used in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined based on the judgment of the practitioner and the circumstances of each patient. The effective dose can be deduced from a dose-response curve derived from an in vitro or animal model test system.

[0052] As a general suggestion, the dose of the antigen-binding polypeptide of the present invention administered to a patient is usually 0.1 mg/kg to 100 mg/kg patient body weight, 0.1 mg/kg to 20 mg/kg patient body weight, or 1 mg/kg to 10 mg/kg patient body weight. In general, due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower dosing dosage and lower dosing frequency of human antibodies are usually possible. Moreover, the administration frequency and dosage of the antibodies of the present invention can be reduced by modification (such as lipidation) to enhance the uptake of these antibodies and tissue penetration (such as, into the brain).

[0053] Methods for the treatment of infections or malignant diseases, disorders or conditions (including administering the antibodies, variants or derivatives of the present invention), before being used in humans, are usually tested in vitro, and then in vivo in acceptable animal models to obtain the desired therapeutic or preventive activity. Suitable animal models (including transgenic animals) are well known to those of ordinary skill in the art. For example, in vitro experiments that demonstrate the therapeutic utility of the antigen-binding polypeptides described herein include the effect assays of the antigen-binding polypeptides on cell lines or patient tissue samples. The effects of the antigen-binding polypeptide on cell lines and/or tissue samples can be determined using techniques known to those skilled in the art, such as tests disclosed elsewhere herein. According to the present invention, in vitro tests that can be used to determine whether a specific antigen-binding polypeptide needs to be used include in vitro cell culture tests in which a patient tissue sample is grown in a culture medium and exposed to or otherwise administered with antibodies and observing the effect of this antibody on the tissue sample.

[0054] In a further embodiment, the composition of the present invention is administered in combination with an antitumor agent, antiviral agent, antibacterial agent or antibiotic preparation or antifungal preparation. Any of these formulations known in the art can be administered in the composition of the present invention.

[0055] In another embodiment, the composition of the invention is administered in combination with a chemotherapeutic agent. The chemotherapeutic agents that can be administered with the composition of the present invention include, but are not limited to, antibiotic derivatives, such as doxorubicin, bleomycin, daunorubicin, defensin; anti-estrogens such as tamoxifen; antimetabolites such as fluorouracil, 5-FU, methotrexate, fluorouridine, interferon .alpha.-2b, glutamic acid, pracamycin, mercaptopurine and 6-mercaptoguanine; cytotoxic agents such as Carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin and vincristine sulfate; hormones such as medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, medroxyprogesterone, diethylstilbestrol diphosphate, chlorinestrol, and testosterone; nitrogen mustard derivatives, such as chlorambucil, chlorambucil, dichloromethyldiethylamine (chlorambucil), thiotepa; steroids such as betamethasone sodium phosphate; and others, such as dacarbazine, asparaginase, mitotane, vincristine sulfide, vinblastine sulfide, and etoposide.

[0056] In another embodiment, the composition of the invention is administered in combination with cytokines. Cytokines that can be administered with the composition of the present invention include but are not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L and TNF-.alpha..

[0057] In other embodiments, the composition of the invention is administered in combination with other therapeutic or preventive therapies (such as radiation therapy).

Pharmaceutical Compositions

[0058] The invention also provides a pharmaceutical composition. Such a composition includes an effective amount of the antibody and an acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, more specifically for human. Further, the "pharmaceutically acceptable carrier" will usually be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of excipient.

[0059] The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which the drug is used. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Salt solutions and aqueous glucose and glycerin solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.

[0060] If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffering agents, such as acetate, citrate or phosphate. It can also be expected to add antibacterial agents such as benzyl alcohol or methyl benzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid and agents for isotonicity adjustment such as chlorine sodium or dextrose. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository using customary binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described by E. W. Martin in Remington's Pharmaceutical Sciences, incorporated by reference. Such a composition will contain a therapeutically effective amount of the antigen-binding polypeptide (preferably in a purified form) and an appropriate amount of carrier so as to provide the patient with an appropriate mode of administration. This formulation should suit the mode of administration. This parental preparation can be contained in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0061] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to routine procedures. Generally, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. The components are usually supplied individually or mixed together in unit dosage form, for example, a lyophilized powder or an anhydrous concentrate in a closed container, for example, an ampoule or sachette indicating the number of active agents. When the composition is administered by infusion, it can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients can be mixed before administration.

[0062] The composition of the present invention can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include salts formed from anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed from cations, such as those derived from sodium, potassium, ammonium, calcium, hydrogen Salts of iron oxide, isopropylamine, triethylamine, ethylhydroxyethylamine, histidine, procaine, etc.

Examples

S-Fab Design and Protein Purification

[0063] The structure of S-Fab is shown in FIG. 1A. The VH-CH1 and VL-CL fragments of anti-CD3 were synthesized and cloned by standard DNA cloning techniques. A signal sequence pelB was added to the N-terminus for periplasmic expression. For site-specific conjugation of PEG, a cysteine residue was added at the C-terminal of light chain followed by a short linker, and then another cysteine (CGGGGC) and a his6 tag. S-Fab was constructed via the heterodimerization of VL-CL/VH-CH1 (anti-CD3 Fab) with anti-CEA VHH nanobody. Flag-tag was added to the C-terminal of heavy chain for detection (FIG. 1B).

[0064] To produce the S-Fab, the two plasmids encoding respective VH--CH--VHH and VL-CL polypeptides were co-transformed into BL21 (DE3, codon plus) competent cells with proper antibiotics. When the absorbance (OD600) of cell culture reached 0.8, 0.2 mM isopropyl-.beta.-d-thiogalactoside (IPTG) was added to induce protein expression. The cells were cultured at 16.degree. C. for another 40 hrs before harvesting. After harvesting the cells by centrifugation, periplasmic extraction was performed by re-suspending the cell pellets 1:4 (g:mL) in a pre-cooled sucrose solution (20 mM Tris-HCl pH 7.5; 25% (w/v) sucrose; 1 mM EDTA). After 15 min incubation on ice, the suspension was centrifuged at 10,000 g for 20 min, and the supernatant fraction was collected as the sucrose fraction. The pellet was then re-suspended in a chilled periplasmic solution (5 mM MgCb) and centrifuged at 10,000 g for 20 min. The supernatant was gathered as the periplasmic fraction.

[0065] The S-Fab protein was then purified from the combined sucrose and periplasmic fractions by a two-step purification: first by the immobilized Ni-NTA affinity chromatography (GE Health, USA), and then by an IgG-CH1 affinity matrix (Lot, 194320005; hermoFisher Scientific Inc, USA) (FIG. 1C). Gel filtration analysis was performed using a Bio-Rad FPLP system and GE Superdex 200.TM. Increase 10/300 GL column with a flow rate of 0.5 ml/min. Fractions (0.5 ml per fraction) were collected, and then subjected to the SDS-PAGE analysis under reducing conditions. The resulting fractions were visualized by the coomassie blue staining. Protein markers (Lot, MWGF200; Sigma Aldrich Co.. Ltd. USA) were loaded as standard controls for gel filtration analysis. Gel filtration analysis showed the intact S-Fab antibodies with molecular weights of .about.130 kD (FIG. 1D), indicated the dimerization of S-Fab (.about.65 kD of monomer), likely formed by the disulfide bond(s) of C-terminal cysteine residues of VL-CL.

Conjugation of S-Fab to PEG (PEGylation)

[0066] The S-Fab was engineered with two terminal cysteine residues located at the C-terminal of CL, which served as the sites for conjugation with a 20 kDa linear MAL-PEG-OMe. S-Fab (approximately 1.35 mg/mL (about 20 .mu.M) in 5.0 mL phosphate buffered saline (PBS, pH 7.4) and three molar equivalents of 1 mM tris (2-carboxyethyl) phosphine (TCEP, final 60 .mu.M, approximately 300 .mu.L) were mixed and incubated for two hours at 22.degree. C. to obtain the reduced S-Fab fragments.

[0067] In order to explore the optimal molar ratio of MAL-PEG-OMe and S-Fab in the PEGylation process, we proceeded a series of reactions with the molar equivalent of PEG:S-Fab as 0:1, 10:1, 20:1, 40:1 and 60:1, respectively (FIG. 2A). MAL-PEG-OMe was dissolved in the sterile water to obtain a working concentration of 20 mg/mL (1 mM). The PEGylation of S-Fab was carried out by mixing the MAL-PEG-OMe (at the working concentration) with reduced S-Fab and shaking at 22.degree. C. for 2 hrs. The resulting samples were subjected to 12% SDS-PAGE electrophoresis (5 ul/sample/PAGE), then for coomassie blue and barium iodide staining of PEG according to reference (FIGS. 2A and 2B). After electrophoresis, the western blot assay was used to detect the PEGylated chain. Briefly, another two gels were transferred to polyvinylidene fluoride membrane (Millipore, USA). After two-hour blocking with 5% skimmed milk, membranes were respectively incubated with mouse monoclonal anti-flag HRP (1:2000, for heavy chain) and mouse monoclonal anti-his IgG (1:3000, for light chain) in 5% skimmed milk. The secondary antibody (goat anti-mouse HRP-conjugated IgG, 1:3000) were incubated with the light chain membrane for another one hour after washing with TBST buffer. The membranes were developed with Pierce's West Pico chemiluminescence substrate (Millipore, USA) after washing with TBST buffer. PEGylated S-Fab was purified using an AKTA avant25 FPLC purification system (GE Healthware, USA) and Superdex 10/300 GL column with a flow rate of 0.8 ml/min. The column was first equilibrated with two column volumes (CV) of distilled water and then two CV of PBS before applying samples. All the collected fractions were analyzed by coomassie blue and barium iodide complex dye after SDS-PAGE at reducing conditions. Fractions for the purified PEGylated S-Fab were pooled together for further studies.

[0068] Increased ratio of PEG:S-Fab in the PEGylation of S-Fab with MAL-PEG-OMe resulted in a higher molecular weight band (.about.107 kD and .about.45 kD), indicating the maleimide functionalized PEG was conjugated to S-Fab. The 45 kD band suggested the single conjugation at the single C-terminal cysteine (FIGS. 2B and 2D). The .about.107 kD and indicates both cysteine residues at the VL-CL terminal were PEGylated (FIGS. 2B and 2D). The .about.107 kD band appeared first and accounts for the majority of light chain, indicating the preferred conjugation on both cysteine residues (FIGS. 2B and 2D). As no decrease of VH--CH-VHH was observed (FIGS. 2A and 2C) and no high molecular weight of VH--CH1-VHH was observed based on western blot (FIG. 2C), suggesting no PEGylation on the VH--CH1-VHH chain. As the band of VL-CL chain was decreased (FIGS. 2A and 2D), and PEGylation corresponds to the VL-CL chain based on anti-his western blot (FIGS. 2B and 2D), it suggested only VL-CL had PEGylation. As the molar ratio of PEG:S-Fab increases, there are also slightly increased higher molecular weight PEGylated bands, suggesting further PEGylation on the other cysteine residues on VH-CH1 or VL-CL. The molar ratio of PEG:S-Fab at 20 was then chosen for conjugation as it had higher VL-CL conjugation without higher molecular weight conjugation.

[0069] To remove the free PEG, free S-Fab, and high molecular weight proteins, the conjugation reaction mixture was subjected to size exclusion analysis. Based on SDS-PAGE followed by coomassie blue staining (FIG. 3B) and barium iodide staining for PEGylation (FIG. 3C), the high molecular weight fraction 1 (FIG. 3A-3C) contained a large proportion of multiple conjugation species. Fraction 2 (FIG. 3A-3C) contains mostly the double and single site conjugated PEG-S-Fab, while fraction 3 (FIG. 3A-3C) containing free S-Fab, free PEG, and single site conjugated S-Fab. Fractions containing mostly the double and single site conjugated PEG-S-Fab (Fraction 2 in FIG. 3) were pooled for further studies.

PEG-S-Fab can bind the tumor antigen CEA and CD3.sup.+ T cells.

[0070] The bispecific S-Fab has two different binding sites, the anti-CEA VHH recognizing CEA on tumor cells, and the anti-CD3 recognizing CD3' on T cells. To check whether PEGylation of S-Fab affects the binding of CEA-positive cancer cells, flow cytometry analysis was performed using LS174T cells, which have over-expression of CEA. CEA-negative cell line SKOV3 was used as negative control.

[0071] Briefly, 1.times.106 (for LS174T and SKOV3) or 5.times.105 (for T cells) cells per sample were collected by centrifugation at 1000 rpm for 5 min and then washed once with 1.0 mL of ice-cold PBS with 0.2% bovine serum albumin (BSA). The primary antibodies, including S-Fab, PEG-S-Fab, and the blank control (Vehicle, PBS only) were added to a final concentration of 10 .mu.g/mL, and then incubated on ice for one hour followed by washing twice with ice-cold PBS with 0.1% BSA. Anti-CD3 FITC (OKT3, final concentration of 10 .mu.g/mL) was used as positive control for CD3.sup.+ antigen binding analysis. Goat anti-human IgG (H+L)-AlexaFluor 488 antibody was then added to a final concentration of 5 .mu.g/mL. The cells were incubated on ice for another hour. After washing the cells twice, flow cytometric detection was then performed.

[0072] Both S-Fab and PEG-S-Fab showed obviously specific fluorescence intensity shifts, suggesting that PEG-S-Fab can still bind to LS174T cells (FIG. 4A). The slight left shift of PEG-S-Fab to the value of S-Fab (FIG. 4B) indicates that the PEGlyation of S-Fab may slightly reduce the binding affinity of S-Fab to its antigen. For SKOV3 cells, both S-Fab and PEG-S-Fab cannot bind the cells due to the lack of CEA antigen on cell surface, suggesting that PEG-S-Fab retains the specific antigen binding (FIG. 4B). Similarly, for the CD3.sup.+ on the T cells, the obvious fluorescence intensity shift of S-Fab and PEG-S-Fab was also presented. PEGylation may also slightly decrease the binding activity to the CD3.sup.+ antigen on T cells (FIG. 4C). The only slightly reduced binding activities of PEG-S-Fab suggested that site specific conjugations in the middle of S-Fab had minimal effects on bispecific antibody binding affinity.

[0073] To further analyze the binding of S-Fab and PEG-S-Fab to cell surface CEA, immunofluorescence assay was performed. Briefly. LS174T and SKOV3 cells (2.5.times.105 cells in 1.0 mL, respectively) were plated on 30-mm confocal glass bottom dishes (NEST, cat. 801002) to 80% confluence. The cells were then washed with cold PBS three times before fixing with 4% paraformaldehyde. The fixed cells were incubated with 20 .mu.g of S-Fab or PEG-S-Fab and then 10 .mu.g goat anti-human IgG (H+L)-AlexaFluor 488 antibody for 2 hour at 4.degree. C. respectively. The cell nuclei were counterstained with DAPI. After washing with PBS, samples were then examined using Olympus FV3000 laser scanning confocal microscope and analyzed by Olympus FV31S-SW_V2.1 software.

[0074] Both S-Fab (upper panel in FIG. 4D) and PEG-S-Fab (upper panel in FIG. 4E) can bind CEA positive LS174T cells. For CEA-positive SKOV3 cells, no binding was observed for S-Fab (lower panel in FIG. 4D) or PEG-S-Fab (lower panel in FIG. 4E), further supporting that PEGlyation maintains the specific binding affinity of S-Fab to its antigen.

PEG-S-Fab has Potent Specific Cytotoxicity Against Tumor Cells

[0075] The CEA-positive human LS174T cells and the CEA-negative human SKOV3 cells were used to assess the in-vitro growth inhibitory effects of S-Fab and PEG-S-Fab. Briefly, LS174T and SKOV3 cells were used as target cells (T), and freshly prepared human CD3.sup.+ T cells without prior stimulation were used as effector cells (E). In vitro cytotoxicity assays were performed in 96-well microplates in triplicates by seeding 5,000 target cells per well in 100 .mu.L of corresponding media. After a 6-hour incubation, an equal volume of CD3.sup.+ T cells were added to each well at an E:T ratio of 10:1, and a series of concentrations (0.033, 0.1, 0.33, 1, 3.3, 10, 33 and 100 nM) of S-Fab or PEG-S-Fab were then added correspondingly. After 72-hour incubation, cell viability was evaluated via a CCK8 assay according to the manufacturer protocol. The absorbance values were detected using a TECAN microplate reader at 450 nm. The survival rate (100%) was calculated as: [(As-Ab)(A0-Ab)].times.100%, where As is the absorbent value of the measurement group, Ab is the absorbent value of medium and A0 is the absorbent value of measurement group at 0 nM.

[0076] Both S-Fab and PEG-S-Fab can kill CEA-positive LS174T cells efficiently even at 0.033 nM (FIG. 5A) while no cytotoxcity against CEA-negative SKOV3 cells (FIG. 5B). The PEG-S-Fab has slightly decreased cytotoxicity than S-Fab (FIG. 5A). This could be due to the slightly decreased antigen binding or steric interference of PEGylation. However, the level of decreased activity seems much lower than many other PEGlyated proteins.

PEGylation Prolongs In Vivo Half-Life of S-Fab

[0077] To determine the circulating half-life of PEG-S-Fab, the intravenous PK profiles of S-Fab and PEG-S-Fab in rat were analyzed. SPF male SD rats (250-300 g) were used for the PK assay. Food was controlled to maintain animals below 350 g in weight. S-Fab (1.0 mg/kg), PEG-S-Fab (1.0 mg/kg) or a volume equivalent of vehicle solution PBS was administered through the caudal vein. The blood sample (each approximately 150-200 .mu.L) was taken from the orbital vein using a capillary under isoflurane anesthesia at 0, 0.5, 1, 2, 4, 8, 16, 24, 36, 48, 72, 96 and 144 hrs after administration. All blood samples were collected into heparinized tubes. Plasma was obtained via centrifugation at 3,500 g for 30 min, and then stored at -80.degree. C. until further analysis.

[0078] S-Fab and PEG-S-Fab in the plasma samples were quantified using an ELISA assay. Briefly, a 100 .mu.L aliquot of 6D6 (mouse anti-human IgG Fab antibody) (1.0 .mu.g/mL in PBS) was coated to each well of a 96-well ELISA microplate (ThermoFisher, USA) for 2 hrs at 37.degree. C. The wells were then washed twice with 200 .mu.L of PBST (PBS+0.05% Tween-20). Wells were then blocked with 200 .mu.L of blocking buffer (PBST containing 1% bovine serum albumin) for 2 hrs at 37.degree. C. Each well was then washed five times with PBST prior to the addition of 100 .mu.L of samples or standards. Samples and standards (100, 80, 50, 40, 30, 20, 10, 5, 1 and 0.1 .mu.g/mL) were prepared in the blocking buffer with the standards (S-Fab) prepared in a 1:10 dilution of plasma using PBS, which was important to avoid matrix effects in the assay. For plasma samples, a 1:3 dilution was used. 100 .mu.L sample or standard aliquots was then added in triplicate and incubated at 37.degree. C. for one hour. Each well was washed again with PBST before the addition of 100 .mu.L of secondary antibody (mouse monoclonal anti-flag mzperoxidase (HRP) antibody at 1:500 dilutions) per well at 37.degree. C. for one hour. After washing for five times, a 100 .mu.L aliquot of TMB substrate solution was added to each well. After 10 min incubation, 100 .mu.L of 2M H.sub.2SO.sub.4 was added to stop the reaction. The absorbance was then detected at 450 nm using a TECAN ELISA microplate reader. Serum elimination t.sub.1/2 and clearance were calculated by 3p97 pharmacokinetic software using standard formula. The results are expressed as the mean.+-.SEM, and comparisons between the groups were made with an unpaired Student's t-test. Differences were considered to be statistically significant if p<0.05.

[0079] S-Fab and PEG-S-Fab were quantified using the ELISA method as described in the part of 210. Quantification of a series of standards showed that the standard curve was y=0.0934.times.+0.0748 (0.ltoreq.x.ltoreq.25 .mu.g/mL) with R.sup.2 value of 0.9982 (FIG. 6A). Based on serum protein concentration up to 144 hrs, the S-Fab had an in-vivo half-life of approximately 3.0 hrs, similar to reported half-life of other Fab in vivo [34-36]. PEGylation significantly extended the half-life of PEG-S-Fab to 36.0 hrs, a 12-fold increase to S-Fab (FIG. 6B).

[0080] S-Fab and PEG-S-Fab stability was assessed in human fresh plasma over two weeks. Briefly. S-Fab and PEG-S-Fab were diluted with human fresh plasma (without platelet), which generated an initial concentration of 100 .mu.g/mL. The samples were incubated at 37.degree. C. for two weeks. At the time intervals of 0, 24, 48, 72, 96, 168, 264 and 336 hr, 40 .mu.L samples were collected and then stored directly at -80.degree. C. till further analysis. The samples were thawed on ice and then centrifuged at 14,000 rpm for 10 mins at 4.degree. C. The supernatant was then subjected to electrophoresis on 12% SDS-PAGE (5 ul sample per well). After electrophoresis, western blot as descripted in 2.4 was performed to analyze the protein level. When incubated with human plasma in vitro at 37.degree. C., the level of S-Fab decreased sharply after 72 hrs (FIGS. 6C and 6E). For PEG-S-Fab, the decrease was much slower (FIGS. 6D and 6F). Thus. PEGylation can also improve the stability of S-Fab in plasma probably due to the protection of enzymatic digestion in plasma.

PEG-S-Fab Induces More Potent In Vivo Anti-Tumor Activity

[0081] The increased in vivo half-life and comparable in vitro cytotoxicity of PEG-S-Fab prompted us to assess the in vivo anti-tumor activity of PEG-S-Fab in an adoptive xenograft model. The in vivo anti-tumor activities of S-Fab and PEG-S-Fab were studied using NOD/SCID mice engrafted subcutaneously with LS174T cells. Briefly, LS174T cells were harvested and washed once with PBS, and then mixed with human PBMCs freshly isolated from healthy donors. The mixtures of 1.times.10.sup.6 LS174T cells and 5.times.10.sup.6 human PBMCs were injected subcutaneously into the right flank of NOD/SCID mice in a total volume of 0.2 mL per mouse. One hour after the engraftment, 0.3 nmol S-Fab (20.0 .mu.g per mouse), and 0.3 nmol PEG-S-Fab (32.0 .mu.g per mouse) or the vehicle control (PBS) were injected intraperitoneally. The animals were then treated daily (0.3 nM per mouse in each group) over the following six days. Tumor volume was measured with calipers in two perpendicular dimensions and was calculated using the formula (width.sup.2.times.length)/2. All data were expressed as the mean SE for each group, and differences between groups were determined by two-way ANOVA using GraphPad Prism 5 software.

[0082] When NOD/SCID mice were transplanted with LS174T cells and fresh isolated human PBMCs, rapid tumor growth was observed. Compared with the vehicle group, significant tumor growth inhibition (p<0.01) was observed when the mice were treated with either S-Fab or PEG-S-Fab in the presence of human PBMCs (FIG. 7). The tumor growth inhibition was more pronounced in the presence of PEG-S-Fab compared to S-Fab (p<0.01), indicating PEGylation of S-Fab protein can enhance the therapeutic efficacy of the S-Fab in the xenograf mouse model.

[0083] All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0084] One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[0085] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0086] Also, unless indicated to the contrary, where various numerical values are provided for embodiments, additional embodiments are described by taking any 2 different values as the endpoints of a range. Such ranges are also within the scope of the described invention.

[0087] When appropriate, the explanatory description herein may be implemented in the absence of any one or more elements, one or more restrictions that are not specifically disclosed herein. Therefore, for example, in each case herein, any one of the terms "including", "substantially consisting of" and "consisting of" can be replaced by the other two terms. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can make modifications and changes to the ideas disclosed herein, and these modifications and changes are still within the scope of the present invention as defined in the appended claims.

Sequence CWU 1

1

261120PRTArtificial Sequenceanti-CEA VHH 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Glu1 5 10 15Ser Leu Thr Leu Ser Cys Thr Ser Ser Thr Leu Thr Phe Thr Pro Tyr 20 25 30Arg Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45Ala Asp Ile Ser Ser Gly Asp Gly Arg Thr Thr Asn Tyr Ala Asp Phe 50 55 60Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Asn Thr Val65 70 75 80Phe Leu Arg Met Thr Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Asn Thr Phe Val Ser Phe Val Gly Ile Ala Arg Ser Trp Gly Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115 1202102PRTArtificial Sequenceanti-CD16 VHH 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Phe Pro Gly Ser Ile Phe Ser Leu Thr 20 25 30Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Thr 35 40 45Ser Ala Thr Pro Gly Gly Asp Thr Asn Tyr Ala Asp Phe Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Ser Ile Ile Tyr Leu Gln65 70 75 80Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 85 90 95Arg Thr Arg Asn Trp Gly 1003102PRTArtificial Sequenceanti-CD16 VHH 3Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr 20 25 30Asn Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ser Ile Thr Trp Ser Gly Arg Asp Thr Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Asn Pro Trp Pro 1004102PRTArtificial Sequenceanti-CD16 VHH 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu1 5 10 15Ser Leu Thr Leu Ser Cys Val Val Ala Gly Ser Ile Phe Ser Phe Ala 20 25 30Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45Arg Ile Gly Ser Asp Asp Arg Val Thr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ile Lys Arg Thr Ala Gly Leu Gln65 70 75 80Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95Gln Thr Asp Leu Arg Asp 1005102PRTArtificial Sequenceanti-CD16 VHH 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Val Ala Ala Gly Ser Ile Phe Thr Phe Ala 20 25 30Met Ser Trp Tyr Arg Gln Ala Pro Arg Lys Glu Arg Glu Leu Val Ala 35 40 45Arg Ile Gly Thr Asp Asp Glu Thr Met Tyr Lys Asp Ser Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Arg Thr Ala Gly Leu Gln65 70 75 80Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95Arg Thr Asp Tyr Arg Asp 1006123PRTArtificial Sequenceanti-Her2 sdAb 6Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Ala Asn Ile Tyr Val Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Val Lys Leu Gly Phe Ala Pro Val Glu Glu Arg Gln Tyr Asp Tyr 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 1207128PRTArtificial Sequenceanti-EGFR1 VHH 7Met Ala Glu Val Gln Gln Ala Ser Gly Gly Gly Leu Val Gln Ala Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Glu Thr Thr 20 25 30Ser Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45Val Ala Gln Ile Ser Ala Ser Gly Leu Gly Ile Asn Tyr Ser Gly Thr 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ala Asp Lys Thr Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Ala Gly Phe His Tyr Ile Ala Ala Ile Arg Arg Thr Thr Asp 100 105 110Phe His Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Arg 115 120 1258121PRTArtificial Sequenceanti-F4+ETEC bacteria VHH 8Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Asn Val Asp Arg Ile Asp 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45Gly Tyr Ile Ser Glu Gly Gly Ile Leu Asn Tyr Gly Asp Phe Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Ser Asn Leu Lys Ser Glu Asp Thr Gly Val Tyr Phe Cys Ala 85 90 95Ala Ser His Trp Gly Thr Leu Leu Ile Lys Gly Ile Glu His Trp Gly 100 105 110Lys Gly Thr Gln Val Thr Val Ser Ser 115 1209127PRTArtificial Sequenceanti-PS2-8 VHH 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Arg Asp 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Val Val 35 40 45Ala Ala Ile Asn Leu Asn Gly Gly Arg Thr Tyr Ser Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Ser Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Glu Gly Asp Val Gly Leu Val Ser Tyr Lys Arg Ser Ser 100 105 110Asn Tyr Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 12510121PRTArtificial Sequenceanti-Huntavirus VHH 10Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Leu Val Gln Ala1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Ser 20 25 30Met Tyr Ser Met Val Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ala Gly Ile Ile Trp Thr Ser Ser Leu Thr Tyr Tyr Ala Asp 50 55 60Ser Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr 85 90 95Tyr Cys Ala Ala Asp Thr Lys Thr Gly Gly Gly Gly Tyr Glu Tyr Trp 100 105 110Gly Gln Val Thr Val Thr Val Ser Ser 115 12011119PRTArtificial Sequenceanti-Huntavirus VHH 11Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser 20 25 30Ser Asp Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Leu Val Ala Phe Ile Thr Asp Asp Gly Gly Thr Asn Tyr Ala Asp Ser 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val65 70 75 80Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Asn Ala Arg Tyr Tyr Ser Gly Gly Tyr Arg Asn Tyr Trp Gly Gln 100 105 110Val Thr Val Thr Val Ser Ser 11512115PRTArtificial Sequenceanti-CD16 VHH 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Phe Pro Gly Ser Ile Phe Ser Leu Thr 20 25 30Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Thr 35 40 45Ser Ala Thr Pro Gly Gly Asp Thr Asn Tyr Ala Asp Phe Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Ser Ile Ile Tyr Leu Gln65 70 75 80Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 85 90 95Arg Thr Arg Asn Trp Gly Thr Val Trp Gly Gln Gly Thr Gln Val Thr 100 105 110Val Ser Ser 11513129PRTArtificial Sequenceanti-CD3 VHH 13Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 20 25 30His Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45Ala Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Asn Thr Met Ser65 70 75 80Leu Gln Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Thr Thr Pro Thr Glu Lys Gly Ser Ser Ile Asp Tyr Trp Gly Gln Gly 100 105 110Thr Gln Val Thr Val Ser Ser Gly Arg Tyr Pro Tyr Asp Val Pro Asp 115 120 125Tyr14654DNAArtificial Sequenceanti-CD19Fab light chain-nucleic acidVL(1)..(336)CL(337)..(654) 14gatattcaga tgactcagag cccttcctca ctgtccgcca gcgtcgggga tcgggtcact 60attacatgca aagcaagcca gagcgtcgac tatgatgggg actcctatct gaactggtac 120cagcagaagc caggcaaagc tcccaagcgg ctgatctacg acgcatcaaa tctggtgagc 180ggcgtccctt ccagattctc tgggagtgga tcaggcacag attttaccct gacaattagc 240tccctgcagc ctgaggactt cgccacttac tattgccagc agagcacaga agatccatgg 300acttttggcg ggggaaccaa agtggagatc aagagaacgg tggctgcacc atctgtcttc 360atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 65415218PRTArtificial Sequenceanti-CD19Fab light chain - amino acidVL(1)..(109)CL(110)..(218) 15Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Arg Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Thr 85 90 95Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 21516666DNAArtificial Sequenceanti-CD19Fab heavy chain-nucleic acidVH(1)..(372)CH(373)..(666) 16caggtgcagc tggtccagtc cggggcagaa gtgaagaaac caggagcctc tgtgaaagtc 60agttgtaagg cttcaggcta taccttcatc tcttactgga tgaactgggt gagacaggca 120ccaggacagg gactggagtg gatgggacag atttggcctg gcgatgggga caccaactat 180aatcagctgc tgaaggataa agcaactctg accgccgaca agagcgcctc caccgcttac 240atggagctgt ctagtctgcg aagcgaagac acagctgtgt actattgcgc acggagagaa 300accacaactg tgggccgcta ctattacgca atggattact ggggacaggg cacactggtg 360actgtctcaa gcgctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 420agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 480gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 660aaagtt 66617222PRTArtificial Sequence4. anti-CD19Fab heavy chain-amino acidVH(1)..(127)CH(128)..(222) 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gln Leu Leu 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210 215 22018639DNAArtificial Sequenceanti-CD20Fab light chain-nucleic acidVL(1)..(321)CL(322)..(639) 18cagatcgtgc tgagccagag ccccgccatc ctgagcgcca gccccggcga gaaggtgacc 60atgacctgca gggccagcag cagcgtgagc tacatccact ggttccagca gaagcccggc 120agcagcccca agccctggat ctacgccacc agcaacctgg ccagcggcgt gcccgtgagg 180ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcagggt ggaggccgag 240gacgccgcca cctactactg ccagcagtgg accagcaacc cccccacctt cggcggcggc 300accaagctgg agatcaagag gacggtggct gcaccatctg tcttcatctt cccgccatct 360gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600agctcgcccg tcacaaagag cttcaacagg ggagagtgt 63919213PRTArtificial Sequenceanti-CD20Fab light chain-amino acidVL(1)..(107)CL(108)..(213) 19Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25

30His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 21020654DNAArtificial Sequenceanti-CD20Fab heavy chain-nucleic acidVH(1)..(360)CH(361)..(654) 20caggtgcagc tgcagcagcc cggcgccgag ctggtgaagc ccggcgccag cgtgaagatg 60agctgcaagg ccagcggcta caccttcacc agctacaaca tgcactgggt gaagcagacc 120cccggcaggg gcctggagtg gatcggcgcc atctaccccg gcaacggcga caccagctac 180aaccagaagt tcaagggcaa ggccaccctg accgccgaca agagcagcag caccgcctac 240atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc caggagcacc 300tactacggcg gcgactggta cttcaacgtg tggggcgccg gcaccaccgt gaccgtgagc 360gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 65421218PRTArtificial Sequenceanti-CD20Fab heavy chain-amino acidVH(1)..(122)CH(123)..(218) 21Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110Ala Gly Thr Thr Val Thr Val Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210 21522639DNAArtificial Sequenceanti-Her2Fab light chain-nucleic acidVL(1)..(321)CL(321)..(639) 22gatattcaga tgacccagag cccgagcagc ctgtccgcaa gcgtcggcga ccgcgtcacg 60attacctgcc gtgcaagcca ggatgtcaac accgcagtgg cttggtatca gcaaaaaccg 120ggtaaagcgc cgaaactgct gatttatagt gcctcctttc tgtactctgg cgtgccgagt 180cgtttctcag gttcgcgcag cggcaccgat tttaccctga cgatcagctc tctgcagccg 240gaagacttcg ccacgtatta ctgccagcaa cattatacca cgccgccgac ctttggtcaa 300ggcacgaaag tcgaaattaa aacggtggct gcaccatctg tcttcatctt cccgccatct 360gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600agctcgcccg tcacaaagag cttcaacagg ggagagtgt 63923213PRTArtificial Sequenceanti-Her2Fab light chain-amino acidVL(1)..(107)CL(108)..(213) 23Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 21024651DNAArtificial Sequenceanti-Her2Fab heavy chain-nucleic acidVH(1)..(357)CH(358)..(651) 24gaagtgcagc tggttgaatc tggcggtggc ctggttcaac cgggtggctc cctgcgtctg 60tcatgtgcag catcgggttt caacattaaa gatacctata tccactgggt tcgtcaggca 120ccgggtaaag gcctggaatg ggtcgctcgc atttacccga ccaatggtta tacgcgttac 180gcggatagcg tgaaaggccg cttcaccatc agcgcagaca cctctaaaaa cacggcttat 240ctgcagatga actcgctgcg tgcggaagat acggccgttt attactgtag ccgctggggt 300ggcgacggct tttacgcaat ggattactgg ggtcaaggca cgctggtcac ggtctcagct 360agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt t 65125217PRTArtificial Sequenceanti-Her2Fab heavy chain-amino acidVH(1)..(119)CH(120)..(217) 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val 210 215266PRTArtificial Sequencelinker with two C-terminal cycterine 26Cys Gly Gly Gly Gly Cys1 5

Claims

1. A PEGylated bispecific antibody, comprising (a) an antigen-binding fragment (Fab) having a light chain variable region (VL) and a light chain constant region (CL), a heavy chain variable region (VH) and a part of a heavy chain constant region (CH1); (b) a single domain antigen-binding fragment (VHH) fused to a C-terminus of the part of the heavy chain constant region (CH1) of the antigen binding fragment (Fab); and (c) polyethylene glycol (PEG) fused to a C-terminus of the light chain constant region (CL) of the antigen-binding fragment (Fab).

2. The PEGylated bispecific antibody of claim 1, wherein the polyethylene glycol is connected to the C-terminus of the light chain constant region (CL) through at least one cysteine residue at the C-terminus of the light chain constant region (CL).

3. The PEGylated bispecific antibody of claim 2, wherein when the polyethylene glycol is connected to the C-terminus of the light chain constant region (CL) through two cysteine residues at the C-terminus of the light chain constant region (CL), a linker sequence is spaced between the cystine residues.

4. The PEGylated bispecific antibody of claim 1, wherein the molecular weight of the polyethylene glycol is 10,000 to 30,000.

5. The PEGylated bispecific antibody of claim 4, wherein the molecular weight of the polyethylene glycol is 20,000.

6. The PEGylated bispecific antibody of claim 1, wherein the antigen-binding fragment is specific for tumor antigens, and the single domain antigen-binding fragment is specific for immune cells.

7. The PEGylated bispecific antibody of claim 6, wherein the tumor antigen is selected from a group consisting of CEA, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, Mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, .alpha.V.beta.3, .alpha.5.beta.1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1 TRAILR2, RANKL, FAP and Tenascin.

8. The PEGylated bispecific antibody of claim 7, wherein the tumor antigen is CEA or Her2.

9. The PEGylated bispecific antibody of claim 6, wherein the single domain antigen-binding fragment is specific for mammalian T cells or mammalian NK cells.

10. The PEGylated bispecific antibody of claim 6, wherein the single domain antigen binding fragment has specificity for any antigen selected from the group consisting of CD3, CD16, CD19, CD28 and CD64.

11. The PEGylated bispecific antibody of claim 10, wherein the antigen is CD16 or CD3.

12. An engineered bispecific antibody comprising (a) an antigen-binding fragment (Fab) having a light chain variable region (VL) and a light chain constant region (CL), a heavy chain variable region (VH) and a part of a heavy chain constant region (CH1); and (b) a single domain antigen binding fragment (VHH) fused to the C-terminus of the part of the heavy chain constant region (CH1) of the antigen binding fragment (Fab); wherein the C-terminus of the light chain constant region (CL) of the antigen-binding fragment (Fab) is engineered to have one or two cysteine residues.

13. The engineered bispecific antibody of claim 12, wherein when there are two cysteine residues, there is a linker sequence between the two cysteine residues.

14. (canceled)

15. A pharmaceutical composition comprising the bispecific antibody of claim 1 and a pharmaceutically acceptable carrier.

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